The number of larvae hatching in each pan was recorded as a measure of fertility. Wing-length Measurement. the bloodmeal to reach the midgut epithelium. After traversing this tissue, the parasites rest beneath the basal lamina and form oocysts, within which thousands of sporozoites develop. Once matured, sporozoites exit oocysts and travel through the mosquito open circulatory system to reach the salivary glands from which they can be released during a subsequent blood meal. Parasite resistance genes should be designed to encode products that inhibit parasite development without having major fitness effects on the mosquito host Bromocriptin mesylate (3). Single-chain antibodies (scFvs) are promising candidates due to their specificity, efficacy, and small size. Transgenic expressing the scFvs m1C3, m4B7 or m2A10, produce significantly fewer parasites than controls when challenged with (6). The m1C3 and m4B7 scFvs were derived from monoclonal antibodies that bind the ookinete proteins Chitinase 1 and Pfs25, respectively. The m2A10 scFv binds the circumsporozoite protein (CSP), the predominant surface protein of sporozoites. An additional feature of m4B7 and m2A10 is the joining of the cecropin A peptide to the scFvs by a polypeptide linker. Cecropin has microbiocidal activity against both bacteria and species (7), and the resulting scFv-peptide proteins could exert both parasite-binding and antimicrobial activity. We posited that a mosquito expressing two scFvs that target different life stages would completely inhibit parasite development. We tested this hypothesis using site-specific recombination to produce strains expressing dual transgenes comprising either m1C3 or m4B7 linked to m2A10. ((site in the transgene-bearing plasmid recombines with an site (docking site) in the mosquito genome (12). Studies of the demonstrated that the strength of transgene expression in different tissues varies among docking sites (13). recipient lines carrying one to three copies of the docking site, four of which were analyzed previously and shown to have no significant fitness load (14). A mutated challenge experiments. No sporozoites could be detected Bromocriptin mesylate in experiments with mosquitoes expressing m1C3 and m2A10 at relevant developmental stages. These studies support the use of dual scFv transgenes as effector molecules in population replacement strategies to control malaria parasite transmission. Results Assembly, Site-Specific Integration, and Expression of the m4B7/m2A10 Transgene. The pBacDsRed-m4B7/m2A10-plasmid was constructed using the AgCPA-m4B7 and AsVg1-m2A10 cassettes assembled previously (Fig.?1) (6). The pBacDsRed vector expresses DsRed, a fluorescent marker distinguished easily from the cyan fluorescent protein (CFP) expressed by recipient-line mosquitoes (16). An sequence inserted into the left-hand terminal repeat DNA of the transposon (pBac LH) allows transgene recombination and insertion at the Cecropin A epitope-tag gene (E tag-m4B7-CecA) is flanked by epitope-tag gene regulatory sequences (CP 5, CP 3). The m2A10 scFv Cecropin A gene (CecA-m2A10-E tag) is flanked by gene regulatory sequences (VG 5, VG 3). Arrows denote the direction of transcription. The scFv transgenes are joined to the DsRed plasmid containing the pUC18 vector, LH and RH sequences joined to a CFP transformation marker and a phage attachment site (and sites forms right and left attachment Rabbit Polyclonal to STK36 sites (and plasmid was microinjected with mutated 30, 43, and 44 (Table?1) (10). This plasmid also was microinjected into 20 and 19A embryos with wild-type integrase mRNA. All docking-site lines except 30 yielded recombinant offspring with 19A, 43, and 44 producing seven, two, and five lines, respectively. Individual DsRed-positive mosquitoes from lines containing multiple docking site transgenes (19A, 43, 44) were outcrossed to wild-type (non-transgenic) mosquitoes to establish independent lines. DsRed-positive individuals from line 20, containing one docking site, were intercrossed to establish a single transgenic line. Table 1. Summary of results of pBacDsRed-scFv-plasmid microinjections into transgenic lines lineDocking sitesIntegrasePlasmidEmbryos injectedAdultsG0 pools /*G1 positive larvae?G1 negative larvae?Positive G0 pools?Transgenic lines20 were intercrossed to form a single transgenic Bromocriptin mesylate line. Fluorescent hybridization in situ and gene amplification (inverse PCR) were used to characterize the docking-site insertions in DsRed-positive 44 individuals (Fig.?S1). Hybridization of a CFP-specific probe to polytene chromosomes revealed three 44 docking sites, designated 44-A,.
